Plasmids for chromosomal recombination of Escherichia coli

ABSTRACT

A temperature-sensitive plasmid which is capable of autonomous replication in  Escherichia coli  K-12 at 10–30° C., but, at a temperature of 33° C. or more, is incapable of autonomous replication in  Escherichia coli  K-12 or is distributed unhomogeneously upon the cell division of  Escherichia coli  K-12, thereby not to be stably carried within cells of  Escherichia coli  K-12 under said temperature, and which is incapable of autonomous replication in a microorganism belonging to the genus  Escherichia  other than  Escherichia coli  K-12 or is distributed unhomogeneously upon cell division of said microorganism at any temperature, thereby not to be stably carried within cells of said microorganism.

TECHNICAL FIELD

The present invention relates to a plasmid to be used for integratinggenes into a microorganism belonging to the genus Escherichia other thanEscherichia coli K-12, a method for integrating the genes into thechromosome, a recombinant strain constructed according to the method,and a method for producing useful substances by using the recombinantstrain.

BACKGROUND ART

When a microorganism engineered to intracellularly carry a plasmid thatexpresses a specific gene is utilized for the production of a usefulsubstance, over-expression of the gene and instability of the plasmidoften cause problems. To solve the problems, modifications of genes on achromosome are carried out as an effective method. Methods involvingmutagenic treatments have long been carried out as a chromosomalengineering technique in Escherichia coli. This method is directed tothe selection of a desired mutant strain from randomly mutated strains,and requires a great deal of work. In addition, deliberate or rationalmanipulation is almost impossible.

On the other hand, P1 transduction using P1 phage is known to be themost versatile technique to deliberately and rationally manipulate thechromosome of Escherichia coli [Zinder, N. D. and Lederberg J., J.Bacteriol., 64, 679 (1952)].

Chromosomal manipulation techniques other than P1 transduction areroughly classified into 2 types.

One type is directed to a method which comprises inserting a gene ofinterest into a plasmid capable of autonomous replication inmicroorganisms other than Escherichia coli but incapable of autonomousreplication in Escherichia coli and transforming Escherichia coli withthe plasmid to obtain a strain in which the gene of interest isintegrated into its chromosome according to the principle of homologousrecombination [A. Chen et al., J. Bacteriol., 176, 1542 (1994)].However, this method involves a drawback that a desired chromosomalrecombinant strain is obtained only at a very low frequency because aplasmid which has been prepared by using a microorganism other thanEscherichia coli as a host cell, is decomposed by restriction enzymeswithin Escherichia coli.

The other type is directed to the method in which a plasmid beingcapable of autonomous replication under the normal growth conditions inEscherichia coli K-12 but incapable of autonomous replication undercertain conditions such as a high temperature condition is used and agene of interest is integrated into a chromosome in accordance with theprinciple of homologous recombination [T. Hashimoto, and M. Sekiguchi,J. Bactereiol., 127, 1561 (1976)].

Escherichia coli that have been widely used in the research andindustrial areas include several types such as the K-12, B, and Wstrains. Many of the genetic recombination techniques have beendeveloped by using the K-12 strain.

Escherichia coli W is suitable for the production of useful substancessuch as amino acids, etc. [S. Furukawa et al., Appl. Microbiol.Biotechnol., 29, 253 (1988)] and serves many uses in fermentativeproduction as it assimilates sucrose and has been actually subjected tohigh density cell culture successfully [I. E. Gleiser and S. Bauer,Biotechnol. Bioeng., 23, 1015 (1981)].

The above-mentioned genetic engineering techniques relate to the K-12strain and no report has so far been made that concerns withmicroorganisms belonging to the genus Escherichia of the types,different from K-12.

DISCLOSURE OF THE INVENTION

The object of the present invention is to establish a new chromosomalrecombination technique in microorganisms belonging to the genusEscherichia other than the K-12 strain and solve the above problems.

The present invention relates to the following (1)–(14).

-   (1) A temperature-sensitive plasmid which is capable of autonomous    replication in Escherichia coli K-12 at 10–30° C., but at a    temperature of 33° C. or more, is incapable of autonomous    replication in Escherichia coli K-12 or is distributed    unhomogeneously upon the cell division of Escherichia coli K-12,    thereby not to be stably carried within cells of Escherichia coli    K-12 under said temperature, and which is incapable of autonomous    replication in a microorganism belonging to the genus Escherichia    other than Escherichia coli R-12 or is distributed unhoinogeneously    upon cell division of said microorganism at any temperature, thereby    not to be stably carried within cells of said microorganism.-   (2) The plasmid according to the above (1), wherein the    microorganism belonging to the genus Escherichia other than    Escherichia coli K-12 is Escherichia coli W or Escherichia coli B.-   (3) The plasmid according to the above (1) or (2), wherein the    plasmid is a plasmid containing a DNA fragment capable of undergoing    homologous recombination with the chromosome of Escherichia coli.-   (4) The plasmid according to any one of the above (1) to (3),    wherein the plasmid is carried by Escherichia coli DH5α/pMTS11910    (FERM BP-6904) or Escherichia coli DH5α/pMTS11914 (FERM BP-6905).-   (5) A plasmid having one or more genes integrated therein, which is    obtainable by integrating any genes into the plasmid according to    any one of the above (1) to (4).-   (6) A method for integrating one ore more genes, which comprises    introducing the plasmid according to any one of the above (1) to (5)    to a microorganism belonging to the genus Escherichia other than    Escherichia coli K-12.-   (7) The method for integrating one or more genes according to the    above (6), wherein the microorganism belonging to the genus    Escherichia other than Escherichia coli K-12 is Escherichia coli W    or Escherichia coli B.-   (8) The method for integrating one or more genes according to the    above (6) or (7), wherein the integration of the genes is an    integration genes in which the plasmid is integrated into a    chromosome.-   (9) The method for integrating one or more genes according to any    one of the above (6) to (8), wherein the integration of one or more    genes is an integration in which a DNA fragment on the plasmid is    substituted with a DNA fragment on a chromosome by homologous    recombination.-   (10) A transformant obtainable by the method according to any one of    the above (6) to (9).-   (11) The transformant according to the above (10), wherein the    transformant is a transformant selected from the group consisting of    Escherichia coli DH5α/pMTS11910 (FERM BP-6904), Escherichia coli    DH5α/pMTS11914 (FERM BP-6905), Escherichia coli WLA-131 (FERM    BP-6902), and Escherichia coli WL-1133 (FERM BP-6903).-   (12) A method for producing a useful substance, which comprises    culturing the transformant according to the above (10) or (11) in a    medium, allowing the useful substance to produce and accumulate in a    culture and recovering the useful substance from the culture.-   (13) The method according to the above (12), wherein the useful    substance is selected from the group consisting of amino acids,    organic acids, nucleic acids, nucleic acid-related substances,    sugars, lipids, vitamins, and pigments, and derivatives thereof.-   (14) The method for producing a useful substance according to the    above (12), wherein the useful substance is a protein.    [1] Mutagenic Treatment of Plasmid

As the plasmid, pMW119 (available from Nippon Gene Co., Ltd.) or aplasmid having the same ori region and the rep gene as those of pMW119is used. An ori region is a region containing the initiation site ofreplication and the rep gene is a gene encoding replicase.

Mutagenic treatment of the plasmid is carried out according to a knownmethod using hydroxylazine [G. O. Humphreys et al., Mol. Gen. Genet.,145, 101 (1976)], etc.

For example, when hydroxylamine is used, the plasmid can be mutated bydissolving about 10 μg of the plasmid in phosphate buffer [50 mmol/lNaH₂PO₄, 1 mmol/l EDTA2Na, pH 6.0 (NaOH)] containing 0.4 mol/lhydroxylamine hydrochloride, and heating the solution at 75° C. for 30to 60 minutes.

(2) Preparation of Temperature-sensitive Plasmids

A plasmid showing temperature-sensitivity in Escherichia coli K-12 canbe obtained by a known method [T. Hashimoto, and M. Sekiguchi, J.Bacteriol., 127, 1561 (1976)].

More particularly, Escherichia coli K-12 is transformed with the plasmidto which a mutagenic treatment has been given to obtain a strain havingresistance to a drug such as ampicillin within the range of 10–32° C.While any method of transformation that can transform Escherichia coliK-12 may be used, electroporation [W. J. Dower et al., Nucleic AcidsRes., 16, 6127 (1988)] that offers high transformation efficiency ispreferred.

Escherichia coli K-12 to which a transformation treatment has been givenis spread on LB agar medium [1.0% Bacto Tryptone (Difco), 0.5% YeastExtract (Difco), 1.0% NaCl 2% agar] containing a drug to be used as amarker and cultured at 10–32° C. for 6–24 hours.

Strains whose growth is confirmed by the culturing are selected astransformants. The transformants are cultured on an agar medium withoutcontaining any drug added at a temperature of 33° C. or more at whichthe transformants can grow.

After the culturing, strains grown are spread on an agar mediumcontaining a drug such as ampicillin and cultured at a temperature of33° C. or more at which the strains can grow for 6–24 hours. Strainscorresponding to those that can not grow under these conditions areseparated from the above agar medium without containing any drug addedand cultured again on the medium without containing any drug added.After the culturing, a plasmid is separated from the strains accordingto a conventional method. The plasmid is the temperature-sensitiveplasmid.

[3] Preparation of Plasmids Incapable of Autonomous Replication in theCells of a Microorganism Belonging to the Genus Escherichia Other ThanEscherichia coli K-12

The plasmid of the present invention, that is, the plasmic incapable ofreplication in the cells of a microorganism belonging to the genusEscherichia other than Escherichia coil K- 12, can be selected fromplasmids showing temperature sensitivity in Escherichia coli K-12, whichare obtained according to the method described in (2) above.

The microorganisms belonging to the genus Escherichiaother thanEscherichia coli K-12 herein may be any microorganisms that belong tothe genus Escherichia other than the K-12 type strain. Examples thereofinclude microorganisms belonging to Escherichia coli of such types asEscherichia coli W, Escherichia coli B, Escherichia coli C, Escherichiacoli 15, etc. and Escherichia coli W or Escherichia coli B is preferred.

More particularly, transformation of a microorganism belonging to thegenus Escherichia other than Escherichia coli K-12 is carried out byusing the plasmid that shows temperature sensitivity in Escherichia coliK-12 to obtain transformants. If the transformants are unable to grow atany temperature when they are cultured in a medium containing a drug,such temperature-sensitive plasmid is the plasmid having the property ofthe present invention.

[4] Integration of a Gene into a Chromosome

The plasmid which can not replicate in a microorganism belonging to thegenus Escherichia other than Escherichia coli K-12 at any temperatureobtained in accordance with the above-described method (hereinafterreferred to as the plasmid of the present invention) is used forintegration into a chromosome.

First, a gene is cloned to the plasmid of the present invention. Thegene may be any gene that participates in the production of a desireduseful substance and has a sequence homologous to a gene of interest inthe chromosome of a microorganism belonging to the genus Escherichiaother than Escherichia coli K-12. The useful substance may be any usefulsubstance so far as the gene participates in the production thereof. Forexample, amino acids such as leucine, etc., organic acids such asisocitric acid, etc., nucleic acids or nucleic acid-related substancessuch as flavin adenine dinucleotide, etc., sugars such as fructose,etc., lipids such as phospholipid, glycolipid, etc., vitamins such asbiotin, etc., and pigments such as carotene, etc., derivatives thereof,proteins such as enzymes encoded by the gene, etc. may be mentioned butthe useful substances are not limited to these substances.

Cloning of the gene can be carried out from Escherichia coli K-12according to a known method [Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1989)] (hereinafterreferred to as Molecular Cloning, Second Edition). Furthermore, it ispossible to artificially modify the nucleotide sequence of the clonedgene according to a known method (Molecular Cloning, Second Edition).For the cloning of the gene and modification of the cloned gene,Escherichia coli K-12 is used as a host cell and culturing is carriedout at 32° C. or less.

The thus constructed recombinant plasmid is used to transform amicroorganism belonging to the genus Escherichia other than Escherichiacoli K-12 and the strain is cultured on a medium containing a drug suchas ampicillin to obtain a drug resistance strain, whereby chromosomalrecombinant strain in which plasmid containing the gene of interest hasbeen integrated into the chromosome can be obtained. To transform themicroorganism, any of known methods such as electroporation, calciumchloride method (Molecular Cloning, Second Edition), etc. can be used.

It is also possible to obtain a strain in which the gene cloned on theplasmid is substituted with the gene originally carried by amicroorganism belonging to the genus Escherichia other than Escherichiacoli K-12 on its chromosome.

More particularly, a strain transformed with the plasmid of the presentinvention is cultured on a medium without containing a drug such asampicillin. Either a solid agar medium or a liquid medium withoutcontaining agar may be used for the culturing so far as thetransformants can grow thereon.

The cultured cell is properly diluted with sterilized physiologicalsaline, spread on LB agar medium without containing a drug such asampicillin, and cultured. Each of the colonies grown is spread on anagar medium containing a drug such as ampicillin and the strainscorresponding to the strains that have not grown are separated from theoriginal medium without containing a drug. From the separated strains,the strain in which the gene on the plasmid of the present invention andthe gene originally present on the chromosome of a microorganismbelonging to the genus Escherichia other than Escherichia coli K-12 aresubstituted by homologous recombination can be selected.

The chromosomal recombinant strain in which the plasmid containing agene of interest is integrated into its chromosome or the strain inwhich the gene originally carried by a microorganism belonging to thegenus Escherichia other than Escherichia coli K-12 is substituted withthe gene cloned on the plasmid is hereinafter referred to as themicroorganism of the present invention.

By using the microorganism of the present invention, useful substancescan be produced.

In the above method, if the integration of the plasmid on the chromosomeis designed so that the site of integration is in the nucleotidesequence of a target gene, it is possible to disrupt the target gene byinserting a new sequence to the target gene. The gene disruptionaccording to this method has an advantage of higher reliability ascompared with inactivation of a gene by mutation.

For the culturing of the microorganism of the present invention, any ofnatural media and synthetic media may be used so far as it is a mediumthat enables efficient culturing of the microorganism of the presentinvention which contains carbon sources, nitrogen sources, inorganicsalts, etc. which can be assimilated by the microorganism of the presentinvention.

As the carbon sources, any carbon sources that can be assimilated by themicroorganism of the present invention can be used. Examples of suitablecarbon sources include carbohydrates such as glucose, fructose, sucrose,molasses containing them, starch and starch hydrolyzate; organic acidssuch as acetic acid and propionic acid; and alcohols such as ethanol andpropanol.

As the nitrogen sources, ammonia, ammonium salts of various organic orinorganic acids such as ammonium chloride, ammonium sulfate, ammoniumacetate and ammonium phosphate, and other nitrogen-containing compoundscan be used as well as peptone, meat extract, yeast extract, corn steepliquor, casein hydrolyzate, soybean cake, soybean cake hydrolyzate, andvarious fermented microbial cells and digested products thereof.

Examples of the inorganic salts include potassium dihydrogenphosphate,dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate andcalcium carbonate.

Culturing is carried out under aerobic conditions, for example, byshaking culture or submerged spinner culture under aeration.

The suitable culturing temperature is 15–40° C. and the culturing periodis usually 16 hours to 7 days. The pH is maintained preferably at3.0–9.0 during the culturing. The pH adjustment is carried out using anorganic or inorganic acid, an alkali solution, urea, calcium carbonate,ammonia, etc.

If necessary, antibiotics such as ampicillin, tetracycline, etc. may beadded to the medium during the culturing.

By the culturing in the above manner, a useful substance can beaccumulated in the culture. After the completion of culturing, theuseful substance can be recovered from the culture by removingprecipitates such as the cell and using a combination of various methodssuch as ion exchange treatment, condensation, salting-out, etc.

When the useful substance is a protein, the protein can be isolated andpurified by conventional methods for isolating and purifying proteins.For example, when the protein is expressed in a soluble form in thecells, after the completion of culturing, the cells are recovered bycentrifugation and suspended in an aqueous buffer, followed bydisruption using a sonicator, French press, Manton Gaulin homogenizer,Dynomill or the like to obtain a cell-free extract. A purified proteincan be obtained from the supernatant obtained by centrifuging thecell-free extract, by using conventional methods for isolation andpurification of proteins such as extraction with a solvent, salting-outwith ammonium sulfate, etc., desalting, precipitation with an organicsolvent, anion exchange chromatography using resins such asdiethylaminoethyl(DEAE)-Sepharose and DIAION HPA-75 (Mitsubishi Kaseicorporation), cation exchange chromatography using resins such asS-Sepharose FF (Pharmacia), hydrophobic chromatography using resins suchas butyl Sepharose and phenyl Sepharose, gel filtration using amolecular sieve, affinity chromatography, chromatofocusing,electrophoresis such as isoelectric focusing, or the like alone or incombination.

When the protein is expressed as an inclusion body in cells, the cellsare similarly recovered and disrupted, followed by centrifugation toobtain the inclusion body of the protein as a precipitate fraction. Theinclusion body of the protein recovered is solubilized with aprotein-denaturing agent. The solubilized protein solution is diluted ordialyzed to reduce the concentration of the protein-denaturing agentcontained in the solubilized protein solution, whereby the normal stericstructure of the protein is restored. Then, a purified proteinpreparation can be obtained through the same isolation and purificationprocedures as mentioned above.

Examples of the present invention are shown below. These examples arenot to be construed as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a restriction map of pLEUR12 containing leuoperon and distribution of each gene in the leu operon presumed from theinformation on Escherichia coli K-12.

FIG. 2 is a diagram showing a restriction map of pTS14-LEUR122containing leu operon and distribution of each gene.

BEST MODES FOR CARRYING OUT THE INVENTION

Unless otherwise noted, the operations in the following examples werecarried out according to the description of Molecular Cloning, SecondEdition.

EXAMPLE 1

Preparation of Plasmid Which is not Maintained in Escherichia coli W

A typical plasmid vector of Escherichia coli, pMW119 (product of NipponGene Co., Ltd.), was subjected to an in vitro mutagenic treatment. Themutagenic treatment was carried out according to the following methodusing hydroxylamine described in G. O. Humphreys et al., Mol. Gen.Genet., 145, 101 (1976).

About 10 μg of pMW119 prepared by ultracentrifugation was dissolved inphosphate buffer [50 mmol/l NaH₂PO₄, 1 mmol/l EDTA2Na, pH 6.0 (NaOH)]containing 0.4 mol/l hydroxylamine hydrochloride. The solution washeated at 75° C. for 40 min. Thereafter, the treated DNA wasprecipitated by the addition of ethanol and dissolved in TE solution [10mmol/l tris(hydroxymethyl)methane, 1 mmol/l EDTA2Na, pH 8.0 (HCl)].

Escherichia coli K-12 DH5α (Bethesda Research Laboratories) wastransformed using the DNA solution. The strain was cultured on LB agarmedium [1% Bacto Tryptone (Difco), 0.5% yeast extract, 0.5% NaCl, 2%agar] containing 50 mg/l ampicillin at 30° C. and about 1700 strainsgrown were selected.

The selected strains were replicated on LB agar medium withoutcontaining ampicillin and cultured at 42° C. The colonies grown wereagain replicated on LB agar medium containing ampicillin and cultured at42° C. and 22 strains that could not grow were selected from theoriginal plate.

By using the plasmid extracted from the selected strains, Escherichiacoli W (ATCC-11105) and Escherichia coli B (ATCC-11303) were transformedby electroporation [William J. Dower et al., Nucleic Acids Research, 16,6127 (1988)]. In this case, the transformation efficiency was 7×10⁸cells/μg of pBR322.

In each of the W and B strain, two kinds of plasmids by whichtransformants showing resistance to ampicillin were obtained at afrequency of 1/10⁷ or less in comparison to the frequency with pMW119 toobtain such transformants were respectively named pMTS11910 andpMTS11914. Escherichia coli DH5α was transformed with pMTS11910 orpMTS11914. The transformants, Escherichia coli DH5α/pXTS11910 andEscherichia coli DH5α/pMTS11914, have been deposited under the BudapestTreaty with National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary: Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan (former Agency of IndustrialScience and Technology, National Institute of Bioscience andHuman-Technology: 1-1-3 Higashi, Tsukuba, Ibaraki, Japan) as of Sep. 30,1999 as FERM BP-6904 and FERM BP-6905, respectively.

EXAMPLE 2

Construction of Temperature-sensitive Plasmid Containing Leu Operon

As a gene to be subjected to chromosomal gene integration, leu operon ofan L-leucine producing Escherichia coli strain (FERM BP-4704)(JapanesePublished Unexamined Patent Application No. 70879/96) was used.Chromosomal DNA of the FERM BP-4704 strain was extracted according tothe method of Saito et al. [H. Saito and K. Miura, Siochem. Biophys.Acta, 72, 619 (1963)] and the chromosomal DNA was partially decomposedwith restriction enzyme Sau3AI. On the other hand, pMW218 (Nippon GeneCo., Ltd.) was decomposed with restriction enzyme BamHI and treated withalkali phosphatase. The DNA solutions thus treated were mixed andsubjected to ligation reaction with T4 DNA ligase.

By using the DNA undergoing the ligation treatment, an L-leucinerequiring Escherichia coli strain, CV437 [M. G. Davis and J. M. Calvo,J. Bacteriol., 129, 1078 (1977)], was transformed by electroporation. Asa result, 66 strains showing kanamycin resistance and L-leucinenon-requirement were obtained. The L-leucine non-requiring strains wereobtained by selecting strains that could grow when cultured on M9minimal medium without containing L-leucine.

From these strains, 12 strains were selected at random and the plasmidDNA was extracted. By using the plasmid DNA, an L-leucine requiringstrain, CV524 [M. G. Davis and J. M. Calvo, J. Bacteriol., 129, 1078(1977)] was transformed by electroporation and the plasmid from whichL-leucine non-requiring strains were obtained was named pLEUR12. Sincethe CV437 strain is deficient in leu A and the CV524 strain in leu D, itwas presumed that leu operon at least containing leu A and leu D wasinserted to pLECR12. Furthermore, a restriction map of the DNA fragmentinserted to pLEUR12 as prepared coincides with the map corresponded withthat of leu ABCD gene of Escherichia coli K-12 whose nucleotide sequencehas already been determined [T. Ura et al., Nucleic Acids Research, 20,3305 (1992)].

The structure of pLEUR12 presumed is shown in FIG. 1.

The cloned leu operon was inserted to pMTS11914 according to thefollowing procedures.

First, pLEUR12 was cleaved with restriction enzymes EcoRI and XbaI, thecleaved DNA fragments were subjected to agarose gel electrophoresis, andan about 9 kb fragment containing leu operon was extracted from the gel.

Separately, pMTS11914 was cleaved with restriction enzymes EcoRI andXbaI and further treated with alkali phosphatase. The treated DNAsolutions were mixed and subjected to ligation reaction with T4 DNAligase. By using the DNA undergoing the ligation treatment, an L-leucinerequiring Escherichia coli strain, C600 [Brenner S., and J. R. Beckwith,J. Mol. Biol., 13, 629 (1965)], was transformed by electroporation. As aresult, about 500 ampicillin resistance strains were obtained. Fromthese ampicillin resistance strains, L-leucine non-requiring strainswere selected in accordance with the method described above, andcultured at 42° C. and strains showing sensitivity to ampicillin wereselected. Plasmid was extracted from the selected strains, the cleavagepattern of the plasmid with restriction enzymes was examined and theplasmid to which the desired leu operon was inserted was namedpTS14-LEUR122 (FIG. 2).

EXAMPLE 3

Integration of pTS14-LEUR122 into the Chromosome of Escherichia coli W

Escherichia coli W 113-3 [ATCC-11105, J. Bcteriol., 60, 17 (1950)] wastransformed with pTS14-LEUR122 by electroporation. Whether or not theampicillin-resistant strains obtained by the transformation had theL-leucine productivity was examined by the bioassay method.

More particularly, the ampicillin-resistant strains obtained were spreadon M9 minimal agar medium (5 g/l glucose, 6 g/l Na₂HPO₄, 3 g/l KH₂PO₄,0.5 g/l NaCl, 1 g/l NH₄Cl, 1 mmol/l MgSO₄, 0.1 mmol/l CaCl₂, 20 mg/lDL-methionine, 2% agar) containing the above-described L-leucinerequiring strain CV524 at a concentration of about 1×10⁷ cells/ml andcultured at 37° C. for 12 hours.

Around the strains having L-leucine productivity, the CV524 strain wasgrown and a white-turbid circle (hereinafter called halo) was formed.Strains forming a halo bigger than the average size of the halos formedwere separated and cultured on LB agar medium containing ampicillin at42° C. Among these strains, those showing resistance to ampicillin wereselected as strains in which pTS14-LEUR122 was integrated into thechromosome and one of such strains was named Escherichia coli WLA-131.

It was confirmed by southern hybridization that pTS14-LEUR122 wasintegrated into Escherichia coli WLA-131 on its chromosome by homologousrecombination.

Escherichia coli WLA-131 has been deposited under Budapest Treaty withNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary: Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan (former Agency of Industrial Scienceand Technology, National Institute of Bioscience and Human-Technology:1-1-3 Higashi, Tsukuba, Ibaraki, Japan) as of Sep. 30, 1999 as FERMBP-6902.

EXAMPLE 4

Preparation of a Strain Having Substitution from the ChromosomallyIntegrated Strain

Escherichia coli WLA-131 was inoculated into 5 ml of LB medium andcultured with shaking at 33° C. for 24 hours. The resulting culture (100μl) was again inoculated into 5 ml of LB medium and cultured withshaking at 33° C. for 24 hours.

The culture was properly diluted and spread on LB agar medium.

The colonies grown were replicated on LB agar medium containing orwithout containing ampicillin to select ampicillin-sensitive strains.L-leucine productivity of the ampicillin-sensitive strains was examinedaccording to the bioassay method described in Example 3 and one of thestrains that formed a halo was named Escherichia coli WLA-1133.

Escherichia coli WLA-1133 has been deposited under the Budapest Treatywith National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary: Tsukuba Central 6, 1-1-1Higashi, Tsukuba, lbaraki, Japan (former Agency of Industrial Scienceand Technology, National Institute of Bioscience and Human-Technology:1-1-3 Higashi, Tsukuba, Ibaraki, Japan) as of Sep. 30, 1999 as FERMBP-6903.

To confirm that the gene on the chromosome of Escherichia coli WLA-1133was recombined as intended, analysis by southern hybridization wascarried out. That is, pLEUR12 was digested with HindIII and BamHI toobtain a 4.4 kb fragment containing leu ABCD. The fragment was modifiedwith digoxigenin and used as a probe. Chromosomal DNA was extracted fromEscherichia coli W113-3, Escherichia coli WLA-131 and Escherichia coliWL-1133, decomposed with SacII and KpnI and subjected to agarose gelelectrophoresis.

After the electrophoresis, the separated DNA was transferred to anitrocellulose membrane and subjected to hybridization with the probe,followed by color development operation. In Escherichia coli W113-3 andEscherichia coli WL-1133 which are the parent strains, only an about 17kb fragment containing leu ABCD was detected. On the other hand, inEscherichia coli WLA-131, two 11 kb and about 19 kb fragments weredetected. pTS14-LEUR122 which was used for integration into thechromosome contains one KpnI site in its multi-cloning site. Therefore,if leu operon on the plasmid was integrated on the chromosome byhomologous recombination as we presumed, two 19 kb and 11 kb fragmentsshould be detected. Thus, it could be confirmed that Escherichia coliWLA-131 carries pTS14-LEUR122 integrated on leu operon on its chromosomeand that in Escherichia coli WL-1133, the integrated plasmid is missingby repeated homologous recombination.

That is, pMTS11914-derived region of pTS14-LEUR122 integrated on thechromosome is missing from the chromosome and one copy of leu operon ispresent on the chromosome of Escherichia coli WL-1133.

EXAMPLE 5

Amino Acid Production Test

Escherichia coli W113-3, Escherichia coli WLA-131, and Escherichia coliWL-1133 were respectively inoculated into 20 ml of a seed medium (2%glucose, 1% peptone, 1% yeast extract, 0.25% NaCl, pH 7.0) in a 250-mlErlenmeyer flask and cultured with shaking at 30° C. for 16 hours. Theobtained seed cultures (2.5 ml each) were respectively inoculated into25 ml of a production medium (3% glucose, 1.6% ammonium sulfate, 0.1%potassium dihydrogenphosphate, 0.2% corn steep liquor, 150 mg/lDL-methionine, 4% trimagnesium phosphate, 1% calcium carbonate, pH 7.0)in a 250-ml Erlenmeyer flask and cultured with shaking at 30° C. for 48hours. Accumulation of L-leucine in the culture after the completion ofculturing was quantitatively determined by high performance liquidchromatography.

The results are shown in Table 1.

TABLE 1 Strain Leu (mg/l) Escherichia coli W113-3 0 Escherichia coliWLA-131 151 Escherichia coli WL-1133 145

INDUSTRIAL APPLICABILITY

By using a plasmid which is capable of autonomous replication inEscherichia coli K-12 but incapable of autonomous replication inEscherichia coli other than Escherichia coli K-12 (microorganismsbelonging to the genus Escherichia other than Escherichia coli K-12), itis possible to integrate genes of interest in any region on thechromosome of microorganisms belonging to the genus Escherichia otherthan Escherichia coli K-12 or to effectively modify genes of interest onthe chromosome.

1. A temperature-sensitive plasmid (i) which is obtainable from a plasmid carried by Escherichia coli DH5 α/pMTS11910 (FERM BP-6904) or Escherichia coli DH5 α/pMTS11914 (FERM BP-6905), (ii) which autonomously replicates in Escherichia coli K-12 at 10–30° C., (iii) at a temperature of 33° C. or more, is incapable of autonomous replication or is distributed unhomogeneously upon cell division of Escherichia coli K-12, and so is not stably carried within Escherichia coli K-12, and (iv) does not autonomously replicate or is distributed unhomogeneously in an Escherichia other than Escherichia coli K-12 at any temperature, and so is not stably carried within said Escherichia other than Escherichia coli K-12.
 2. The plasmid according to claim 1, wherein the Escherichia other than Escherichia coli K-12 is Escherichia coli W or Escherichia coli B.
 3. The plasmid according to 2, wherein the plasmid contains a DNA fragment capable of undergoing homologous recombination with the chromosome of Escherichia coli.
 4. A plasmid, comprising the plasmid according to any one of claims 1 to 3, said plasmid having one or more genes integrated therein.
 5. A method for integrating, which comprises introducing the plasmid according to claim 4 to a microorganism belonging to the genus Escherichia other than Escherichia coli K-12, wherein said one or more genes are integrated into said microorganism belonging to the genus Escherichiaother than Escherichia coli K-12.
 6. The method for integrating one or more genes according to claim 5, wherein the Escherichia microorganism other than Escherichia coli K-12 is Escherichia coli W or Escherichia coli B.
 7. The method for integrating one or more genes according to claim 5, wherein the plasmid is integrated into a chromosome.
 8. The method for integrating one or more genes according to claim 7, wherein a DNA fragment on the plasmid is substituted with a DNA fragment on a chromosome by homologous recombination.
 9. A transformant obtained by the method according to claim
 7. 10. The transformant according to claim 9, wherein the transformant is a transformant selected from the group consisting of Escherichia coli DH5α/pMTS11910 (FERM BP-6904), Escherichia coli DH5α/pMTS11914 (FERM BP-6905), Escherichia coli WLA-131 (FERM BP-6902), and Escherichia coli WL-1133 (FERM BP-6903).
 11. A method for producing a useful substance, which comprises culturing the transformant according to claim 9 in a medium, allowing the useful substance to produce and accumulate in a culture and recovering the useful substance from the culture.
 12. The method according to claim 11, wherein the useful substance is selected from the group consisting of amino acids, organic acids, nucleic acids, nucleic acid-related substances, sugars, lipids, vitamins, and pigments, and derivatives thereof.
 13. The method for producing a useful substance according to claim 1, wherein the useful substance is a protein.
 14. A plasmid, comprising the plasmid according to any one of claims 1 to 3, said plasmid having one or more genes integrated therein.
 15. A method for integrating which comprises introducing the plasmid according to claim 14 to Escherichia coli W or Escherichia coli B, wherein said one or more genes are integrated into said Escherichia coli W or Escherichia coli B.
 16. The method for integrating one or more genes according to claim 6, wherein the plasmid is integrated into a chromosome.
 17. The method for integrating one or more genes according to claim 15, wherein the plasmid is integrated into a chromosome.
 18. The method for integrating one or more genes according to claim 17, wherein a DNA fragment on the plasmid is substituted with a DNA fragment on a chromosome by homologous recombination.
 19. A method for producing a useful substance, which comprises culturing the transformant according to claim 10 in a medium, allowing the useful substance to produce and accumulate in a culture and recovering the useful substance from the culture.
 20. The method according to claim 19, wherein the useful substance is selected from the group consisting of amino acids, organic acids, nucleic acids, nucleic acid-related substances, sugars, lipids, vitamins, and pigments, and derivatives thereof.
 21. The method for producing a useful substance according to claim 20, wherein the useful substance is a protein.
 22. The plasmid according to any one of claims 1 to 3, wherein the Escherichia other than Escherichia coli K-12 is all Escherichia other than Escherichia coli K-12. 